A SPECIFIC MICROMETHOD FOR THE COLORIMETRIC DETERMINATION OF GLYCINE IN BLOOD AND URINE*

Transcription

1 A SPECIFIC MICROMETHOD FOR THE COLORIMETRIC DETERMINATION OF GLYCINE IN BLOOD AND URINE* BY BENJAMIN ALEX;ASDER, GRETA LANDWEHR,.iSD ARNOLD M. SELIGMAN (From the Medical and Surgical Research Laboratories, Beth Israel Hospital, and the Departments of Medicine and Surgery, Harvard Medical School, Boston) (Received for publication, April 26, 1945) Recently we described a method for the determination of alanine based upon its conversion into acetaldehyde by the action of ninhydrin (1). A method for the measurement of glycine has been devised which involves its conversion by ninhydrin to formaldehyde; the latter is then measured by its reaction with chromotropic acid. Earlier methods for the measurement of glycine are less sensitive and specific than the one here proposed. In 1902 Fischer (2) described a procedure for the gravimetric determination of glycine by isolating it from protein hydrolysates. Other gravimetric methods were devised by Town (3) and Bergmann and Fox (4). The former added nitranilic acid to a protein hydrolysate containing glycine and weighed the insoluble glycoco11 nitranilate thus obtained. The hydrolysate had to be freed of many inorganic salts which interfered with the precipitation. Bergmann and Fox (4) used trioxalatochromiate to produce an insoluble compound with glycine. All of these methods are too insensitive to be applicable to biologic materials in which the amounts of glycine are very small. Zimmermann (5) reported a calorimetric method for glycine based upon its reaction with o-phthalic dialdehyde. The method was subsequently improved by Klein and Linser (6) and Patton (7). Many substances other than glycine, such as histidine, histamine, arginine, cysteine, tryptophane, and ammonia, gave similar reactions and consequently had to be removed before the addition of the dialdehyde. The sensitivity of the method was limited to a minimum of about 1 mg. of glycine. The reaction between a-amino acids and ninhydrin has been clearly * The work described in this paper was done under a contract, recommended by the Committee on Medical Research, between the Office of Scientific Research and Development and Harvard University. It was also aided by a grant from the Josiah Macy, Jr., Foundation. Shortly before this communication was submitted, a paper by Douglas A. MacFadyen appeared in the March issue of the Journal of Biological Chemistry, p. 107, on the measurement of formaldehyde by the use of chromotropic acid. imac- Fadyen defined conditions under which, by action of ninhydrin and chromotropic acid, 0.96 mole of formaldehyde per mole of glycine could be measured. 51

2 52 DETERMINATION OF GLYCINE elucidated (8, 9). Amino acids are deaminated and converted into aldehydes containing 1 less carbon atom. The basis of the method here described for the determination of glycine is its conversion by ninhydrin into formaldehyde which is then measured by allowing it to react with 1,8-dihydroxynaphthalene-3,6-disulfonic acid (chromotropic acid). This reaction, first described by Eegriwe (lo), is used by Boyd and Logan (11) in their method for the measurement of serine. Method Reagents- Ninhydrin solution, 1 per cent (triketohydrindene hydrate, Eastman). Phosphate buffer, ph 5.5 (glass electrode). 3.5 gm. of tripotassium phosphate are added to 100 ml. of a 20 per cent solution of monopotassium phosphate. Sulfuric acid, 0.66 N. Sodium tungstate solution, 10 per cent. Concentrated sulfuric acid, sp. gr Chromotropic acid solution, 5 per cent 1, S-dihydroxynaphthalene-3,6- disulfonic acid, Eastman. The solution is stored in a refrigerator in an all-glass bottle and is stable for at least 2 weeks. Determination of Glycine in Blood-5.0 ml. of a protein-free filtrate of blood, prepared in the usual way (1) with sulfuric acid and sodium tungstate solution, are placed in the flask of a Stotz all-glass still (12) containing 2 ml. of phosphate buffer, 1 ml. of ninhydrin solution, and a glass bead. The flask is attached to the condenser, and the contents are distilled rapidly into a test-tube calibrated to 10.0 ml. After about 7 ml. of distillate have been collected, the flask is cooled to room temperature by immersing it in a water bath, and then disengaged. 2 ml. of distilled water are added, and distillation is continued to dryness. The neck of the still is heated gently with the flame to distil the last few drops which may have condensed there. The total distillation time should not exceed 15 minutes. The receiving test-tube is removed, and enough distilled water is added to make a total volume of 10.0 ml. After the contents are thoroughly mixed, 5.0 ml. are pipetted into an Evelyn (13) tube, and while it is being cooled and agitated in an ice bath, 4.0 ml. of concentrated sulfuric acid are added slowly. When the reaction mixture has returned approximately to room temperature, 3 drops (0.1 ml.) of the chromotropic acid solution are added. The test-tube is shaken, corked lightly, and placed in a boiling water bath for 30 minutes. After the solution is cooled, the intensity of color is measured in an Evelyn or Klett photoelectric calorimeter, with a No. 565 or No. 540 filter. 1 If an Evelyn photometer is not used, a Pyrex test-tube of similar dimensions (15 X 2 cm.) may be substituted.

3 ALEXANDER, LANDWEHR, AND SELIGMAN 53 Determination of Glycine in Urine-5.0 ml. of a 1:50 dilution of urine are handled in the same way as blood filtrate. It is necessary to run a blank by repeating the procedure on another 5.0 ml. aliquot of the diluted urine with the substitution of 1 ml. of distilled water for the ninhydrin solution. DISCUSSION Absorption Curve2 of Colored Compound Produced by Interaction of Chromotropic Acid and Formaldehyde Obtained from Glycine (Fig. l)-the colored compound which results from the reaction between formaldehyde I E;DM CL 3?0 xii coo w FIG. 1. Visible absorption spectrum of the compound resulting from interaction of chromotropic acid and formaldehyde produced from glycine. E is the molecular cxt,inction coefficient; D, optical density; M, molecular weight (glycine); C, concentration of glycine (gm. per liter) ; L, cell length (cm.). and chromotropic acid absorbs maximally light of wave-lengths 330, 342, 478, and 575 mp. The logarithms of the molecular extinction coefficient at these values are 5.678,5.686,3.946, and respectively. As might be expected from the above, a No. 565 filter was found to give maximum sensitivity. Since most photoelectric calorimeters are furnished with No. 540 filters, calibration curves with both filters are presented for the Evelyn and Klett calorimeters (Figs. 2 and 3). Relationship between Color Intensity and Concentration of Formaldehyde 2 We are indebted to Dr. William G. Dauben of Harvard University for his assistance in obtaining t.he absorption curve. A Beckman D. U. photoelectric spectrophotometer was used.

4 54 DETERMINATION OF GLYCINE and Glycine (Figs. 2 and 3)-The intensity of color obtained by allowing formaldehyde3 to react with chromotropic acid was directly proportional to the concentration of the former. This was also true for a pure solution of glycine when subjected to the procedure outlined above. The glycine curve could be superimposed on that obtained for formaldehyde when due allowance was made for stoichiometric difference. Sensitivity of Method-As little as 0.2 y of formaldehyde, representing 0.5 y of glycine, can be measured with the Evelyn photometer. This FIG. 2. Relationship between intensity of color and equivalent amounts of formaldehyde and glycine. 540 mp filter. corresponds to a minimum concentration of glycine in blood of 0.2 mg. per cent. Speci@ty of Method-Eegriwe (10) described the remarkable specificity of the reaction between formaldehyde and chromotropic acid. Among many other naturally occurring substances only a few, such as glyceraldehyde, arabinose, fructose, and cane sugar, give any color. The color obtained from them was yellow, whereas the color from formaldehyde is rose-pink. Furfural in high concentrations also gives a pink color. These substances cannot interfere with the determination of glycine as outlined above, because they are not volatile with steam (with the possible exception of furfuml) mg. of trioxymethylene were dissolved in 1 liter of distilled water. Under the influence of the strong sulfuric acid used in the reaction, trioxymethylene was converted quantitatively to formaldehyde.

5 ALEXANDER, LANDWEHR, AND SELIGMAN 55 Boyd and Logan (11) report that 10 per cent of glucosamine can be hydrolyzed by strong mineral acid to give formaldehyde. Although glucosamine may exist in blood, there is no evidence of its breakdown to yield formaldehyde under the conditions of our method. The distillation of urine alone, however, yields a small amount of formaldehyde, as determined with chromotropic acid. Since glucosamine must be hydrolyzed with 7 N hydrochloric acid for 24 hours before as much as 10 per cent of it is broken down to give formaldehyde, it is unlikely that a 15 minute distil- FIG. 3. Calibration curve for glycine obtained with 565 and 540 ~I-IJI filters with the Evelyn photometer. Correlation between analyses of multiples of blood filtrate, of urine, and of glycine. lation of diluted urine at a ph of 5.5 can result in appreciable amounts of formaldehyde on this basis. No formaldehyde is obtained when the following substances are subjected to the method for glycine determination: aspartic acid, alanine, tyrosine, lysine, leucine, isoleucine, norleucine, tryptophane, ornithine, threonine, phenylalanine, proline, hydroxyproline, methionine, cystine, cysteine, homocysteine, valine, norvaline, diiodotyrosine, arginine, serine, /3- alanine, glutamic acid, cw-aminoisobutyric acid, /3-amino-n-butyric acid, benzoylalanine. Alanylglycine interferes to the extent that 5 equivalent parts are required to give the same color intensity as 1 part of glycine. It is unlikely that our alanylglycine was hydrolyzed as a consequence of the reaction, since

6 56 DETERMINATION OF GLYCINE Material Blood TABLE Duplicability of Glycine Determinations on Blood and Urine - -- Specimen Urine 6 7 No. I Paired glycine determinations Deviation y per ml. y per ml. per cent * 47.2* * 67.1* * 64.4* * 68.0* * 44.8* * 61.6* * 73.2* * 37.3* * 54.1* * 314* * 364* 6 560* 550* 2 Average... 4 * Glycine added. TABLE Duplicability of Glycine Determinations on Aliquots of Same Blood Glycine y per ml II Deviation from mean j3er cent (Mean) h3 (Average) we were unable to detect any alanine in the reaction mixture. Accordingly we suspect that our alanylglycine was contaminated with some glycine. Among criteria commonly used in establishing the specificity of a new

7 ALEXANDER, LANDWEHR, AND SELIGMAN 57 analytical method is that in which measurements of the substance in question are made on multiples of the material to which the method is applied. The results of these determinations should be superimposable on those obtained on multiples of the pure substance when they are plotted on the same scale. The analyses of multiples of blood filtrate and urine (Fig. 3) coincide almost perfectly with determinations on a solution of pure gly tine. Blood Urine TABLE Recover Y 0; f Glycine Added to Blood and Urine III Glycine added* Recovery of added glycine -f pw ml per CCnI * The concentration of glycine in blood and urine was 20 to 30 y and 150 to 240 y per ml. respectively. Duplicability of Method and Recovery of Added Glycine (Tables I to III)- The largest discrepancy between duplicate determinations of glycine was about 16 per cent. Most of the discrepancies fell within 6 per cent and the average deviation was 4 per cent. It was possible to recover 86 to 105 per cent of glycine added to blood or urine in amounts comparable to those originally present Comment If the formaldehyde produced by the action of ninhydrin on the glycine in a protein-free filtrate of blood is not removed rapidly, substantial losses of formaldehyde occur. This may be due to interaction between formaldehyde and other amino acids which may not have been as rapidly converted as glycine into their respective aldehydes. It has been shown (14) that

8 58 DETERMINATION OF GLYCINE formaldehyde exhibits a great affinity for certain amino acids, particularly those with SH or OH groups in the p position. Losses are greater at ph 4 and 7 than at ph 5.5. Our experience with glycine determinations at these ph levels is in accord with these findings. The addition of more water during the distillation is necessary to assure complete recovery of formaldehyde. In dilute solution this substance exhibits very low volatility. This property is the basis for methods of separating other aldehydes by aeration (1, 14) from formaldehyde. In order to avoid losses which may arise in the distillation of volatile aldehydes, the distillate usually is collected into bisulfite. Because of the low volatility of formaldehyde in very dilute solution, it can be distilled without loss when bisulfite is omitted. Since this reagent interferes markedly with the development of color, its omission is advantageous. Certain facts concerning the reaction between chromotropic acid and formaldehyde are noteworthy. Eegriwe (10) and Boyd and Logan (11) state that strong sulfuric acid and heat are necessary to develop the color. They run the reaction in about 75 per cent sulfuric acid and heat for 10 minutes. Eegriwe (10) can detect as little as 1 part of formaldehyde in 250,000. Boyd and Logan (11) can measure about 50 y of formaldehyde in 17 ml. of aqueous distillate (3 parts in 1 million). We have found that within certain limits the concentration of sulfuric acid determines the rate of the reaction between chromotropic acid and formaldehyde. When the ratio of acid to formaldehyde solution is 6: 1, the color appears almost immediately; with a ratio of 2 : 1 the time required to complete the reaction is 10 minutes. 30 minutes are necessary when the ratio is 4:5, and in proportions of 3:5 more than 1 hour is necessary. The intensity of color, however, is not affected. By using a ratio of 4 of acid to 5 of aqueous formaldehyde solution, the sensitivity of the method is increased over that of Eegriwe (10) and Boyd and Logan (11)) since larger amounts of formaldehyde may be measured in a relatively smaller total volume of reaction mixture. In this way and by using an Evelyn photometer one can measure as little as 0.04 y of formaldehyde per ml. (1 part in 25 million). Efforts to increase the sensitivity by using fuming, instead of concentrated, sulfuric acid or by adding inorganic salts of sodium, potassium, iron, nickel, copper, or zinc were unsuccessful. SUMMARY 1. A method for the calorimetric determination of glycine in blood and urine is described. 2. The method is also applicable to the determination of formaldehyde.

9 ALl%XANDER, LANDWEHR, AND SELIGMAN 59 BIBLIOGRAPHY 1. Alexander, B., and Seligman, A. M., J. Biol. Chem., 169, 9 (1945). 2. Fischer, E., 2. physiol. Chem., 36, 227 (1902). 3. Town, B. W., Biochern. J., 30,1833 (1936). 4. Bergmann, M., and Fox, S. W., J. Biol. Chem., 109,317 (1935). 5. Zimmermann, W., 2. physiol. Chem., 189,4 (1930). 6. Klein, G., and Linser, H., Z. physiol. Chem., 206, 251 (1932). 7. Patton, A. R., J. BioZ. Chem., 108,267 (1935). 8. Ruhemann, S., J. Chem. Sot., (1911). 9. Grassmann, W., and von Amim, K., Ann. Chem., 610,288 (1934). 10. Eegriwe, E., Z. anaz. Chem., 110, 22 (1937). 11. Boyd, M. J., and Logan, M. A., J. BioZ. Chem., 148,279 (1942). 12. Stotz, E., J. BioZ. Chem., 148,585 (1943). 13. Evelyn, K. A., J. BioZ. Chem., 116, 63 (1936). 14. Shinn, L. A., and Xcolet, B. H., J. BioZ. Chem., 138, 91 (1941).

10 A SPECIFIC MICROMETHOD FOR THE COLORIMETRIC DETERMINATION GLYCINE IN BLOOD AND URINE OF Benjamin Alexander, Greta Landwehr and Arnold M. Seligman J. Biol. Chem. 1945, 160: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1